Emergence and Evolution of OXA-23-Producing ST46Pas-ST462Oxf-KL28-OCL1 Carbapenem-Resistant Acinetobacter baumannii Mediated by a Novel ISAba1-Based Tn7534 Transposon

Carbapenem-resistant Acinetobacter baumannii (CRAB) isolates of global clone 1 (GC1) and global clone 2 (GC2) have been widely reported. Nevertheless, non-GC1 and non-GC2 CRAB strains have been studied less. In particular, no reports concerning sequence type 46 (ST46Pas) CRAB strains have been described thus far. In this work, the genomic features and possible evolution mechanism of ST46Pas OXA-23-producing CRAB isolates from clinical specimens are reported for the first time. Antimicrobial susceptibility testing of three ST46Pas strains revealed identical resistance profiles (resistance to imipenem, meropenem, ciprofloxacin and the combination of cefoperazone/sulbactam at a 2:1 ratio). They were found to belong to ST46Pas and ST462Oxf with capsular polysaccharide 28 (KL28) and lipooligosaccharide 1 (OCL1), respectively. Whole-genome sequencing (WGS) revealed that all contained one copy of chromosomal blaOXA-23, which was located in a novel ISAba1-based Tn7534 composite transposon. In particular, another copy of the Tn7534 composite transposon was identified in an Hgz_103-type plasmid with 9 bp target site duplications (TSDs, ACAACATGC) in the A. baumannii ZHOU strain. As the strains originated from two neighboring intensive care units (ICUs), ST46Pas OXA-23-producing CRAB strains may have evolved via transposition events or a pdif module. Based on the GenBank database, ST46Pas strains were collected from various sources; however, most were collected in Hangzhou (China) from 2014 to 2021. Pan-genome analysis revealed 3276 core genes, 0 soft-core genes, 768 shell genes and 443 cloud genes shared among all ST46Pas strains. In conclusion, the emergence of ST46Pas CRAB strains might present a new threat to healthcare settings; therefore, effective surveillance is required to prevent further dissemination.


Introduction
Acinetobacter baumannii is an important pathogen that emerged only several decades ago, causing severe nosocomial infections due to its high-level resistance to various antimicrobial compounds, including carbapenems [1,2]. In 2019, the Centers for Disease Control and Prevention (CDC) recognized carbapenem-resistant Acinetobacter as one of the "Urgent Threats" and a top priority because of the limited options for treatment [3]. Carbapenemresistant A. baumannii (CRAB) strains pose a tremendous global health issue, especially for hospitalized patients with immune dysfunction in intensive care units (ICUs) [4].
Thus far, clones that do not belong to GC1 or GC2 CRAB strains have neither been reported nor studied in any detail, with very few exceptions. Moreover, ST46 Pas A. baumannii has been a relatively rare clone until now. Only one investigation from 2019 in Germany reported a carbapenem-susceptible A. baumannii (CSAB) collected from an infected animal (a horse with conjunctivitis), which belongs to ST46 Pas A. baumannii [12]. Researchers only described the draft genome sequence of A. baumannii strain 161514. Based on the Oxford scheme, ST46 Pas A. baumannii strains could be assigned to ST462 Oxf and have a close genetic relationship with ST1333 Oxf strains. However, there have been no related studies concerning ST462 Oxf and ST1333 Oxf clones to date.
In the current study, we characterized genomic features and possible evolutionary events occurring in three ST46 Pas OXA-23-producing clinical CRAB isolates for the first time. The genetic environment of the bla OXA-23 gene was analyzed in detail using a combination of short-read Illumina and long-read MinION whole-genome sequencing (WGS). A novel ISAba1-based transposon (Tn7534) or pdif module may play a key role in the resistance gene transfer of bla OXA-23 from the chromosome to a plasmid with an Hgz_103-type replicon. We also performed comparative genomics of all ST46 Pas strains available in public databases and conducted the pan-genome analysis of the clone for the first time.

Transfer of Carbapenemase Resistance Determinants
Mating assays and chemical transformation were conducted to study the transferability of bla OXA-23 . However, despite several attempts, no transconjugants and transformants were obtained, possibly indicating that the determinants are not readily transferred.
To analyze the KL and OCL of strains, Bautype and Kaptive software were used. The strains DETAB-C9 and DETAB-P65 were found to contain KL28 and OCL1, matching the 100% coverage to the reference sequence with 97.41% and 98.75% identity, respectively. Likewise, the KL and OCL in A. baumannii ZHOU were identical to those in DETAB-C9 and DETAB-P65, with a nucleotide identity of 97.41% and 98.77%. Similarly, identical KL and OCL results were obtained using Kaptive and Bautype.

Chromosome Analysis of ST46 Pas Strains
A hybrid assembly of short reads and long reads was performed to generate the complete genome sequences of A. baumannii strains. All strains exhibited a circular chromosome with a size of approximately 4 Mb and a GC content of 39% ( Table 2). The chromosome of the A. baumannii ZHOU isolate contained a copy of the novel ISAba1-derived Tn7534 transposon. Corresponding to 28 bp site-specific recombinases XerC/XerD (C/D) and XerD/XerC (D/C) (also named pdif ), 20 pdif sites were found in the chromosome of DETAB-C9 and DETAB-P65 strains. However, only 14 pdif sites were identified in the A. baumannii ZHOU chromosome, and pdif 2 and pdif 3 sites at each side of the bla OXA-23 segment (Table S1). In addition, no prophage regions were identified in the chromosome of the three clinical strains.

Genetic Analysis of Plasmids
We identified one plasmid each in DETAB-C9 and DETAB-P65, which we called pDETAB-C9-1 and pDETAB7b, respectively. Based on the analysis of replicons, they both belonged to the Aci6 type with a size of 73,444 bp ( Table 2). A similar plasmid, a 72,233 bp Aci6-type plasmid (called pZHOU-2), was identified in the ZHOU isolate, with 33.50% GC content (Table 2). Interestingly, no resistance genes were found to be encoded in the Aci6-type plasmids ( Figure 1A). In contrast to the other two strains, another Hgz_103-type plasmid was identified in the A. baumannii ZHOU isolate, which we named pZHOU-1. A second copy of the novel ISAba1-based Tn7534 transposon was found in this plasmid, which exhibited the typical 9 bp target site duplications (TSD; ACAACATGC) ( Figure 1B). belonged to the Aci6 type with a size of 73,444 bp ( Table 2). A similar plasmid, a 72,233 bp Aci6-type plasmid (called pZHOU-2), was identified in the ZHOU isolate, with 33.50% GC content (Table 2). Interestingly, no resistance genes were found to be encoded in the Aci6-type plasmids ( Figure 1A). In contrast to the other two strains, another Hgz_103type plasmid was identified in the A. baumannii ZHOU isolate, which we named pZHOU-1. A second copy of the novel ISAba1-based Tn7534 transposon was found in this plasmid, which exhibited the typical 9 bp target site duplications (TSD; ACAACATGC) ( Figure 1B).  were collected from the sputum and rectal swab of patient 1 (upper white cartoon character) on the fourth floor of the ICU. Circular maps of the chromosome and plasmid are shown using black and blue circles, respectively. The position of novel transposon Tn7534 is indicated by the blue box, and its specific structure is drawn with 9 bp target site duplications (TSDs) indicated using different-colored flags based on the TSD sequence. (B) The A. baumannii ZHOU strain was collected from ascites of patient 2 (lower grey cartoon character) on the third floor of the ICU in the same hospital. Circular maps of the chromosome and plasmid are also shown. The Hgz_103-type plasmid is indicated by an orange circle. Two copies of Tn7534 transposon are indicated by a blue box with 9 bp identical TSDs (ACAACATGC) on either side.

Plasmid Comparison and the Evolution of ST46 Pas CRAB Mediated by a Novel Tn7534 Transposon
With 99.99% identity and 94% coverage, the plasmid pZHOU-1 was identical to the A. baumannii ZW85-1 plasmid ZW85p2 (GenBank accession CP006769), first isolated in 2013 in Beijing (Figure 2A,B). However, no oriT or mobile genetic elements (MGEs) were found in the pZHOU-1 plasmid. identical TSDs (ACAACATGC) on either side.

Plasmid Comparison and the Evolution of ST46Pas CRAB Mediated by a Novel Tn7534 Transposon
With 99.99% identity and 94% coverage, the plasmid pZHOU-1 was identical to the A. baumannii ZW85-1 plasmid ZW85p2 (GenBank accession CP006769), first isolated in 2013 in Beijing (Figure 2A,B). However, no oriT or mobile genetic elements (MGEs) were found in the pZHOU-1 plasmid.  We then attempted to verify the evolutionary hypothesis about how the OXA-23producing ST46 Pas CRAB originated. In detail, in step 1, a complete Tn2009 composite transposon harboring bla OXA-23 was inserted into the A. baumannii chromosome to form 9 bp TSDs (AAAATATTT) flanking both sides ( Figure 2C). Following this event, another copy of ISAba1 interrupted the original ISAba1, possibly contributing to the generation of ISAba1∆ and the formation of the novel transposon Tn7534 ( Figure 2D). Eventually, the Tn7534 composite transposon was likely to integrate into the chromosome ( Figure 2E). At one point, which cannot be conclusively determined from the sequence data (in parallel, before or after the genomic integration), the transposon was inserted into the pZHOU1 plasmid ( Figure 2F), causing an 11 bp deletion that led to the formation of the signature 9 bp TSD (ACAACATGC).

Phylogenetic Analysis of All ST46 Pas A. baumannii Strains from NCBI Database
To further analyze the characteristics of ST46 Pas A. baumannii strains, a query using the NCBI GenBank database was performed. We found sequences of eight other ST46 Pas A. baumannii strains. Based on the MLST type of the Oxford scheme, all strains, apart from A. baumannii strain 161514, belonged to ST462 Oxf . According to the previously published data, A. baumannii strain 161514 belonged to ST462 Oxf as well; however, it exhibited a mutation in gdhB (Oxf_gdhB_59 A > T). We then reanalyzed sequences that showed the strain belonged to a new ST Oxf , namely ST2098 Oxf . Isolates were collected in Germany or China from several sources, including rectal swabs, conjunctivitis, sputum, blood and ascites, between 2014 and 2021 ( Figure 3A). The hosts were mainly human, but-as previously mentionedone strain was isolated from a horse (Equus ferus caballus). All ST46 Pas A. baumannii strains, which were collected from patients in Hangzhou (China), contained the bla OXA-23 gene. In contrast, no bla OXA-23 gene was identified in A. baumannii strain 161514 isolated from the equine in Germany. Genetic relationship analysis showed a rather close genetic connection between the two strains, DETAB-C9 and DETAB-P65. Additionally, three other strains (AB_HZ_B30, AB_HZ_S30, AB_HZ_B28) collected from another hospital but the same city, Hangzhou, were closely related.

Pan-Genome and Single Nucleotide Polymorphisms (SNPs) Analysis
SNP analysis data illustrate the large diversity of ST46 Pas A. baumannii strains. The DETAB-C9 and DETAB-P65 strains were found to be closely related, with only a 16-SNP difference observed ( Figure 3B). A total of 18 to 33 SNPs were found in the AB_HZ_B30, AB_HZ_S30 and AB_HZ_B28 strains. In terms of the number of SNPs, there were large differences in the A. baumannii strain 161514 isolated in Germany when compared to the other strains, which were first found in China.
The genomes of all available ST46 Pas A. baumannii strains were re-annotated. We then performed a gene presence/absence analysis. Bioinformatic data revealed 3276 core genes, 0 soft-core genes, 768 shell genes and 443 cloud genes ( Figure S1A). The presence/absence of genes were visualized with the phylogenetic tree ( Figure S1B).

Pan-Genome and Single Nucleotide Polymorphisms (SNPs) Analysis
SNP analysis data illustrate the large diversity of ST46Pas A. baumannii strains. The DETAB-C9 and DETAB-P65 strains were found to be closely related, with only a 16-SNP difference observed ( Figure 3B). A total of 18 to 33 SNPs were found in the AB_HZ_B30, AB_HZ_S30 and AB_HZ_B28 strains. In terms of the number of SNPs, there were large

Discussion
Hospital-acquired infections caused by CRAB strains pose a growing clinical problem that has become a concern worldwide [13 -15]. CRAB strains can persist in the hospital environment outside of patients, while at infection sites, they can form biofilms, further complicating treatment. Especially in the ICU, accidental dissemination can lead to infections in immunocompromised patients admitted due to non-infection-related medical issues [16][17][18].
In this work, we describe the first complete genome sequences of ST46 Pas and ST462 Oxf strains, which were collected from ICU patients in China. In 2019, Wareth et al. described a single draft genome sequence of a CSAB isolate recovered from a horse with conjunctivitis in Germany [12]. To our knowledge, this study is the first comprehensive report of ST46 Pas CRAB strains that also explores possible evolutionary pathways, including the integration and dissemination of composite transposons.
Mobile genetic elements (MGEs), including insertion sequences (ISs), integrons (In) and transposons (Tn), play a crucial role in antimicrobial resistance gene (ARG) transfer among different kinds of pathogens or between the chromosome and plasmids in a strain [20,21]. Here, the IS26 transposase, a member of the IS6 family, has the remarkable capability to spread ARGs across many Gram-negative bacteria [22,23], especially Klebsiella pneumoniae [24] and E. coli [25]. However, in A. baumannii, ISAba1-based composite transposons are associated with carbapenem resistance and their transfer [8]. Here, we delineated the possible evolutionary pathway of the genetic segment containing bla OXA-23 . The segment harboring bla OXA-23 likely emerged and transferred via translocation events of the novel ISAba1-related Tn7534 transposon. Previous studies uncovered that site-specific recombinases XerC/XerD (C/D) and XerD/XerC (D/C) are able to mediate ARG transfer, including that of tet(39) [26], bla OXA-24 [27] and bla OXA-58 [1]. In our study, we identified two C/D and D/C pdif sites near the Tn7534 transposon containing the bla OXA-23 gene. The DNA segment encompassing the C/D and D/C pdif sites, including the Tn7534 transposon, possibly forms a pdif module. Based on this data, we inferred that another possible mechanism of the mobilization of the Tn7534 transposon containing the carbapenemase gene bla OXA-23 was the recombinase proteins XerC and XerD. A previous study revealed that prophage regions occasionally contain different resistance genes in A. baumannii strains, especially for bla NDM-1 and bla OXA-23 , demonstrating an important role of phages in the transfer of ARGs [28]. In contrast to this report, we were unable to identify prophage regions in our three clinical strains. Consequently, phage-mediated transduction of the bla OXA-23 carbapenem resistance gene is unlikely for the strains we investigated.
Of note is the finding that all ST46 Pas CRAB strains were isolated in Hangzhou (China), as far back as 2014. This illustrates that ST46 Pas CRAB strains have existed in China for at least 8 years. Fortunately, its spread to other locations in the world has not been reported thus far. Our comprehensive pan-genome analysis, which highlighted the similarity of the strains based on the shared 3276 core genes, also revealed the diversity of the isolates, which we assessed on the basis of the differences in SNPs.
While our study aimed to characterize ST46 Pas CRAB strains, there were also limitations to our work. One is that we previously found the A. baumannii ZHOU strain to possess a hypermucoviscous phenotype, for which the virulence level could be further assessed [10]. Likely more important is the fact that we failed to establish the transferability of the Hgz_103-type plasmid harboring bla OXA-23 within the Tn7534 transposon via conjugation or chemical transformation. One possible reason for this is that a T4SS-related transfer (tra) system, oriT region and relaxase were not identified in this plasmid, explaining the Antibiotics 2023, 12, 396 9 of 13 failure of our conjugation experiments. With regard to the transformation experiments, the uptake of DNA fragments via natural transformation with fragments larger than 50 kb is a challenge for bacteria [29]. Moreover, co-mobilization with another conjugative plasmid or the transfer via outer membrane vesicles could be explored. The lack of rapid and frequent transfer via any of the above mechanisms might explain the locally contained presence of the strains.

Patient Information, Bacterial Isolation and Identification
DETAB-C9 was isolated from the sputum of an 85-year-old male patient in the fourthfloor ICU of our hospital in Hangzhou, China, on 22 September 2019. This patient underwent screening of an oral swab and rectal swab every week. DETAB-P65 was collected from the rectal swab on 8 October 2019 from the same patient. In brief, the rectal swab was placed in 2 mL of tryptic soy broth (TSB) with 0.1% sodium thiosulfate and then incubated at 37 • C for one day. Then, 20 µL of overnight culture was plated onto Acinetobacter sp. CHROMagar plates (CHROMagar, Paris, France), which were supplemented with 2 mg/L meropenem. After 24 h of static culture at 37 • C, a single colony was chosen according to its color (red) and morphology, then streaked onto a Mueller-Hinton (MH) agar plate (Oxoid, Hampshire, UK), incubated overnight at 37 • C. A single colony was selected. Isolate identification was performed via matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; bioMérieux, Marcy-l'Étoile, France) and further confirmed via 16S rRNA gene-based PCR and sequencing.
A. baumannii strain ZHOU was collected from ascites in the clinical laboratory during the routine diagnostic of another 63-year-old male patient in the third-floor ICU of the same hospital on 2 December 2021.

Conjugation and Chemical Transformation Experiments
To determine the transferable ability of the Hgz_103-type plasmid carrying bla OXA-23 in the A. baumannii ZHOU strain, conjugation experiments using a rifampicin-resistant derivative of A. baumannii ATCC 17978 as the recipient strain were performed using the film mating method [1,18]. Transconjugants were selected on MH agar plates containing rifampicin (50 mg/L) and imipenem (2 mg/L). Donor or recipient bacteria culture alone was used as the control. The identity of transconjugants was confirmed via PCR. Experiments were carried out in triplicate independently.
Chemical transformation assays were further performed when conjugation experiments failed. The plasmid-harboring bla OXA-23 gene was transferred into E. coli DH5α via chemical transformation with imipenem (2 mg/L) for selection [31].

Nucleotide Sequence Accession Numbers
The complete genome assemblies of the chromosome and plasmids from A. baumannii DETAB-C9, DETAB-P65 and ZHOU are available from GenBank under accession numbers CP104295-CP104296, CP077835-CP077836 and CP104297-CP104299, respectively.

Conclusions
This study describes, for the first time, the genomic characteristics of ST46 Pas OXA-23-producing CRAB isolates. Possible evolutionary pathways indicate that the strains emerged via translocation events of the ISAba1-related Tn7534 composite transposon or the pdif module. Thus far, ST46 Pas CRAB strains have been mainly recovered from hospitals in Hangzhou, China. Nonetheless, effective surveillance should be implemented for ST46 Pas CRAB strains to prevent their dissemination and outbreaks in China and globally.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antibiotics12020396/s1. Figure S1. Pan-genome analysis of ST46 Pas A. baumannii strains using Roary. (A) The numbers of genes belonging to core, soft-core, shell and cloud groups are shown as a pie chart. (B) Matrix of presence/absence genes generated by Roary and the phylogenetic tree of 11 ST46 Pas A. baumannii strains. Figure was created using Phandango. Table S1. Site-specific recombinases XerC/XerD (C/D) and XerD/XerC (D/C) in the chromosome of A. baumannii ZHOU isolate.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki and approved by the local Ethics Committee of Sir Run Run Shaw Hospital (approval number 20190802-1).

Informed Consent Statement:
This study was approved by the Sir Run Run Shaw Hospital (SRRSH) local ethics committee, Zhejiang University (approval number 20190802-1) and informed consent was obtained before sampling.